CN111569158B - Preparation method of biomimetic tissue engineering scaffold with photothermal responsive controllable drug release - Google Patents

Abstract

The invention discloses a preparation method of a bionic tissue engineering scaffold with photo-thermal responsiveness and controllable drug release. The method comprises the following specific steps: preparing bioactive nanoparticles; preparing an electrospinning receiving substrate; preparing a high molecular biological material solution, and blending and electrospinning the bioactive nano-particles and the high molecular biological material solution; and stripping the stent to obtain the stent. The stent has excellent biocompatibility, mechanical property, intelligent responsiveness and drug controlled release performance, can accelerate nerve regeneration, and solves the problems of unsatisfactory long-distance nerve defect repair effect and deficient function recovery. Provides ideal material and preparation method for nerve recovery of nerve injury patients.

Description

Preparation method of biomimetic tissue engineering scaffold with photothermal responsive controllable drug release

technical field

The invention belongs to the technical field of biomedical materials, and in particular relates to a preparation method of a biomimetic tissue engineering scaffold with photothermal responsiveness and controllable drug release.

Background technique

Peripheral nerve injury is a common clinical disease. For those with short defect gaps, the defect can be directly repaired with precision sutures or small gap cannulas, etc. However, for long-distance nerve injuries, direct suture is not feasible. At present, with the development of microsurgical equipment and technology, the clinical treatment effect of peripheral nerve injury has been continuously improved. The most commonly used method is bridging with tissue engineered grafts, which has been proved to be an effective method for repairing peripheral nerve injury. However, Compared with autologous nerve grafts, there is still a gap in its repair effect. Therefore, it is urgent to develop new tissue-engineered nerve grafts.

The study found that the graft surface topology plays an important role in regulating cell growth and tissue regeneration. The biomimetic topology on the surface of the scaffold can not only change the morphology and arrangement of cells, but also affect the growth and function of cells. Biomaterials with topological structures are more conducive to the growth of cells, and cells grown on the scaffolds have better orientations according to the topological orientations already formed by the topological structures. The formation of micro-topological structures on nanofibrous scaffolds made by electrospinning has been shown to significantly enable the adhesion, development, growth and differentiation of various cells on tissue engineering scaffolds. Such surface topological scaffolds have also been reported in nerve regeneration, but the role of individual topology in regulating and promoting nerve regeneration is limited.

Bioactive molecules are a class of substances that have different biological functions such as promoting cell adhesion and growth, and can be proteins, polypeptides, growth factors, and so on. The loading of bioactive molecules into grafts has a significant promoting effect on cell and tissue regeneration. At present, there are many types of bioactive molecules for peripheral nerve regeneration, including YIGSR, IKVAV, NGF, bFGF, etc., all of which can effectively promote the process of nerve regeneration. However, after implantation into the body, the bioactive molecules in the graft have problems of poor stability and easy loss, resulting in insufficient supply of nutrients in the later stage, resulting in long-term implantation failure and difficult recovery of nerve function. Therefore, achieving controlled release of bioactive molecules is critical for the success or failure of nerve regeneration grafts.

Smart responsive materials are a class of functional or intelligent polymers that can respond to various external stimuli (electricity, heat, light, sound, and magnetism), such as bending, shrinking, folding, relaxing or crawling. There are many studies on the improvement of tumor treatment efficiency, controlled drug release and in vitro diagnosis of smart responsive materials, but few studies have been reported on combining them with tissue engineered grafts to achieve controlled drug release in grafts. For implantable artificial grafts, intelligent responsive materials can perform remote controlled drug release therapy for specific injury sites, and can also deform under the action of external stimuli to realize remote automatic regulation of the shape and function of the graft. It is convenient to construct grafts with complex three-dimensional morphological structures. This brings convenience to the patient and reduces the risk of secondary surgery. Therefore, smart responsive materials have great potential application value in the field of tissue engineering.

To sum up, it can be seen that the topological structure of the graft surface and the internal loading of bioactive molecules with controlled release will have a significant effect on promoting nerve regeneration. However, there are few reports on the use of smart responsive materials to achieve biomimetic topology of controlled release of bioactive molecules. Research on the use of grafts for peripheral nerve injury repair.

SUMMARY OF THE INVENTION

Purpose of the invention: In view of the existing problems and the deficiencies of the prior art, the technical problem to be solved by the present invention is to construct an artificial nerve graft with an intelligent responsive controlled-release bioactive molecule inside and a biomimetic micro-nano topology on the surface, In order to better realize the function of accelerating nerve regeneration, it can provide a beneficial choice for patients with peripheral nerve injury in clinical practice. To this end, the present invention provides a preparation method of a biomimetic tissue engineering scaffold with photothermal responsiveness and controllable drug release. The surface micro-nano topological electrospinning receiving substrate was prepared by micro-molding technology, and photothermal nanoparticles with good dispersibility and loaded with bioactive biomolecules were obtained after modification of photothermal nanoparticles. The thermal nanoparticles are evenly blended with the polymer biomaterial solution, and the above mixed solution is electrospun onto the electrospinning receiving substrate with a micro-nano topological structure on the surface by electrospinning technology, so as to obtain an intelligent responsive controlled-release biomaterial inside. Artificial nerve grafts with active molecules and surface biomimetic micro-nano topology. The surface of the graft has anisotropic micro-nano topology, which can not only change the shape and arrangement characteristics of nerve cells on the scaffold, but also affect the function of nerve cells. Grafts with topological structures are more conducive to cell migration, guiding nerve cells to grow along existing topological structures, thereby accelerating tissue migration and growth. At the same time, under the irradiation of near-infrared light, the internal photothermal nanoparticles respond to realize the controllable release of the loaded bioactive molecules, which is beneficial to the long-term function of the nerve regeneration graft after implantation and improve the therapeutic effect. The invention controls the concentration of bioactive factors released by photothermal stimulation of nanoparticles and the synergistic effect of anisotropic topology inducing the growth of nerve cells on the surface of the scaffold, which can accelerate nerve regeneration and solve the problem that the current long-distance nerve defect repair effect is not ideal. Restoring the missing problem.

Technical scheme: In order to achieve the above purpose of the invention, the present invention adopts the following technical scheme: The present invention provides a preparation method of a biomimetic tissue engineering scaffold with photothermal responsiveness and controllable drug release, aiming to better regulate the migration of nerve tissue And growth, and improve the long-term effect of nerve regeneration graft to repair peripheral nerve injury, provide an important reference for the clinical treatment and rehabilitation of patients with peripheral nerve injury.

A biomimetic tissue engineering scaffold with photothermal responsive controllable drug release, the scaffold body material is a polymer biomaterial with excellent biocompatibility; the scaffold contains a controlled release biomaterial with a function of promoting nerve regeneration Active molecules, the bioactive molecules are loaded by nanoparticles with photothermal effect; the surface of the scaffold has an anisotropic topology that can regulate cell-oriented growth and migration; surroundings.

As an optimization: the polymer biomaterials with excellent biocompatibility are chitin, chitosan, alginate, collagen, polycaprolactone (PCL) or polylactide (PLA).

As an optimization: the controlled release bioactive molecules for promoting nerve regeneration are polysaccharides, nucleic acids, proteins, lipids, polypeptides or growth factors.

As an optimization: the nanoparticles with photothermal effect are carbon nanotubes, iron tetroxide or gold nanoparticles.

As an optimization: the anisotropic topological structures that can regulate the oriented growth and migration of cells include micro-nano topological structures and regular micro-nano grooves.

As an optimization: the microenvironment in which the scaffold can better bionic nerve regeneration includes glial cells, nerve growth factors, and extracellular matrix.

The preparation method of the biomimetic tissue engineering scaffold with photothermal responsiveness and controllable drug release comprises the following steps:

(1) Preparation of bioactive nanoparticles loaded with bioactive molecules that promote nerve regeneration;

(2) Prepare a polymer biomaterial solution, blend the above bioactive nanoparticles with the polymer biomaterial solution, and then electrospin

(3) using a micro-molding method to prepare an electrospinning receiving substrate with an anisotropic topology on the surface;

(4) The biomimetic topological tissue engineering scaffold with the function of promoting nerve regeneration was prepared by electrospinning.

As an optimization: the method of modifying the photothermal nanoparticles in the step (1): dopamine hydrochloride (DOPA), genipin, or aminopropyl ethoxysilane coupling agent (APTE) can be used to modify the photothermal nanoparticles The particles are coated and modified.

As an optimization: in the step (2), the ratio of the mass volume ratio of the bioactive nanoparticles to the polymer biomaterial solution blending is 1:100 to 1:10000.

As an optimization: the mold used in the micro-molding method in the step (3) includes a PDMS mold or a PMMA mold; the electrospinning receiving substrate can be a natural biological material or a synthetic biological material substrate, and the substrate is imprinted by the micro-molding method onto a coverslip.

Taking the preparation of YR\DFO-Dopa@MWCNT modified PCL/CS scaffolds as an example, the biomimetic topological tissue engineering scaffolds prepared by this method contain the controlled release bioactive molecule Dopa with the function of promoting nerve regeneration and the polypeptide YR. Bioactive molecules (dopamine Dopa, polypeptide YR) are loaded by nanoparticle carbon nanotubes with photothermal effect; the electrospinning receiving plate prepared by micro-molding method is obtained by chitosan artemisia mixed glue embossing, The surface of the stamp has a biomimetic topology structure, so after the electrospinning film of the chitosan artemisia seal is peeled off from the chitosan artemisia seal, the surface of the scaffold can have anisotropic topology; at the same time, this method The prepared biomimetic topological tissue engineering scaffold is prepared from the polymer chemical material polycaprolactone PCL and the natural material chitosan CS. With the help of the polymer chemical material polycaprolactone material, the electrospinning technology has excellent fiber-forming ability, and the fiber filament Uniform thickness, strong mechanical properties, good biocompatibility and other advantages overcome the difficulty of maintaining an effective electrospinning operation when chitosan materials are electrospun alone, resulting in the emergence of chitosan spinning. Poor fiber-forming ability, uneven fiber thickness, and insufficient mechanical properties. At the same time, thanks to the excellent biocompatibility of chitosan, its surface has cell recognition signals and high affinity with nerve cells, which makes the invented PCL-chitosan hybrid scaffold stable in performance and more conducive to the regeneration of peripheral nerve cells.

Specifically include the following steps:

1. Preparation of a nanoparticle loaded with bioactive molecules with photothermal effect, comprising the following steps:

(1) Put 0.24g of Tris into 200ml of Milli-Q and stir well to prepare a Tris solution with pH=8.5. If the pH is lower than 8.5, titrate Tris by titrating NaOH solution to 8.5;

(2) Take dopamine Dopa powder and add it to Tris solution to prepare polydopamine solution, stir well, after the solution turns reddish brown after full stirring, add nanoparticles to polydopamine solution, add 1 ml of polydopamine solution for every 10 mg of nanoparticles, and The reaction was carried out in the dark for 72 h and centrifuged at 10,000 r/min, and then the remaining precipitate was dried in a drying box to obtain Dopa@MWCNT; wherein, the ratio of Dopa and Tris solution was 1 ml of Tris solution. 2mg Dopa to prepare polydopamine solution;

(3) Wrap the container containing the mixed solution with tin foil to protect it from light, poke a few holes with a sharp object at the mouth of the container to ensure that the oxygen in the air can contact the solution, wrap the container containing the mixed solution with tin foil and put it into a mechanical shaker Medium shaking, after shaking, the solution was discarded from the shaker container, the supernatant was taken out, and the precipitate was dried;

(4) Wash the Dopa@MWCNT obtained by drying with Milli-Q, put the cleaned solution into a centrifuge for centrifugation, discard the centrifuged supernatant, and dry the precipitation to obtain Dopa@MWCNT powder;

(5) Add Dopa@MWCNT powder to the mixed solution of DFO solution and YR solution, put the solution into a mechanical shaker to shake, and then take out the solution from the shaker and put it into a centrifuge for centrifugation. The centrifugal speed is 10000r/min, the time is 3min; wherein, the prepared DFO solution concentration is 200μg/ml, the YR solution concentration is 200μg/ml, and the volume ratio of the prepared 200μg/ml DFO solution and 200μg/ml YR solution mixed solution is 1 : 1; A maximum of 10mg Dopa@MWCNT powder can be added to the mixed solution of 1ml of DFO solution and 1ml of YR solution, and the mixed solution needs to be shaken in a mechanical shaker for 6 hours or more to ensure that the oxygen in the air can contact the solution;

(6) Discard the supernatant after centrifugation, and put the remaining precipitate into a drying box to dry to obtain YR\DFO-Dopa@MWCNT.

2. The preparation of chitosan and Artemisia japonica solution includes the following steps:

(1) glacial acetic acid is dissolved in water, and the concentration of glacial acetic acid solution is 3%; wherein, the glacial acetic acid solvent is water, and the volume ratio of glacial acetic acid and water is 3:100;

(2) Configuration of chitosan solution: 3% glacial acetic acid solution is the solvent of chitosan, and the concentration of chitosan solution is 3%. During the configuration process, the chitosan solution just prepared needs to be stirred, and the stirring time is 24h; wherein, the mass-volume ratio of chitosan and glacial acetic acid solution is 3:100;

(3) Add 0.1g Artemisia annua in every 5ml of chitosan solution, and the chitosan and Artemisia annua solution just prepared need to be stirred, and the stirring time is 24h, wherein, the mass-volume ratio of Artemisia annua and chitosan solution is 1: 500;

(4) The prepared solution of chitosan and Artemisia japonica is sealed and stored in a refrigerator at 4 degrees.

3. Preparation of electrospinning receiver plate, including steps as follows:

(1) Use a syringe to drop a drop of chitosan and Artemisia annua solution on a glass disc, and imprint the topologically anisotropic topology hydrogel on the glass disc with the solution to ensure that the anisotropic topology hydrogel is compatible with The solution between the glass discs does not generate bubbles, and the bubbles are squeezed out. The imprinted anisotropic topology hydrogel and glass disc must be left at room temperature for 24 hours; among them, the anisotropic topology structure The topological units of hydrogels are 10 μm, 30 μm or 50 μm, respectively;

(2) Separation of the anisotropic topology hydrogel and glass disc. Put the peeled glass disc into a large dish, and then perform alkali treatment with NaOH solution with a solution concentration of 4%;

(3) Carefully clean the glass disc with Milli-Q for more than three times, until the cleaning solution is detected as neutral by pH test paper. The glass disc after cleaning is dried in a vacuum drying box. After drying, observe under a light microscope to confirm that It should be dried and stored without error; the topology observed under the light microscope should not have a large number of small bubbles. If a large number of small bubbles appear, it means that the above alkali treatment steps are not completely sufficient, and further alkali treatment should be performed;

(4) Cover the aluminum foil on the cardboard to make the surface of the receiving board have electrical conductivity. The conductive material is aluminum foil, which is used to connect the electrodes on the back, and paste it on the front of the receiving board to dry and preserve it with complete, clear, A glass disc with anisotropic topology without a lot of small air bubbles, this receiving plate is the receiving end of the electrospinning machine.

4. Using electrospinning technology to prepare a biomimetic tissue engineering scaffold with photothermal responsiveness and controllable drug release, the steps are as follows:

(1) 10wt% PCL solution is prepared, and each 1g PCL and 10ml hexafluoroisopropanol can be taken out and stored after shaking and stirring at room temperature for 1 d; wherein, the mass volume ratio of PCL and hexafluoroisopropanol solution is 1:10;

(2) The CS solution of 2wt% is prepared, and each 0.2g CS and 10ml of hexafluoroisopropanol can be taken out and stored after being shaken and stirred for 1 d; wherein, the mass volume ratio of CS and hexafluoroisopropanol is 1:50;

(3) Mix 2wt% CS solution and 10wt% PCL solution, mix 5ml PCL solution and 200μL CS solution, the mixing volume ratio of PCL solution and CS solution is 25:1, and the solution is mixed at room temperature Carry out, stirring time is 1d;

(4) Mix the prepared PCL/CS solution with YR\DFO-Dopa@MWCNT powder, wherein the mass volume ratio of YR\DFO-Dopa@MWCNT powder and PCL/CS mixed solution is controlled at 0.01-5mg/ between the mL range;

(5) Take the above mixed solvent and put it into the electrospinning launch end syringe as the launch end; the glass disc with anisotropic topology on the surface is used as the receiving end of the electrospinning, and electrospinning is carried out after adjusting the parameters. The distance between the needle and the receiving plate is 15cm, the voltage during electrospinning is 20.0kv, and the speed during electrospinning is 0.160ml/h. The needle selected for electrospinning should be a 19 gauge needle, and the electrospinning time is 5h. The temperature is set to room temperature;

(6) Take off the spun receiving plate with the support, and place it in a glass dish with distilled water to peel off the spinning membrane support. The process of peeling and spinning should be carried out in water. It is easy to be involved in the air and destroy the topology structure. The wire is to separate the bracket from the glass wafer of the receiving plate, turn out the topological structure and place it on a new transparent glass slide. The peeled bracket should be dried in a drying box and stored. Glass wafers with topological structure should be stored in a drying box after drying. After drying, glass wafers with topological structure can be recycled for many times if the topological structure is not destroyed.

Technical effect: Compared with the prior art, the present invention has the following advantages.

1. For the first time, intelligent responsive biomaterials were developed and used to construct nerve regeneration grafts to achieve the treatment and repair of nerve tissue damage, which significantly improved the success rate of tissue engineered nerve grafts in the treatment of nerve injury patients.

2. For the first time, photothermal nanoparticles loaded with bioactive molecules and biomimetic functional topology were combined to construct a biomimetic tissue-engineered nerve graft with controllable release of bioactive molecules. Real-time remote control of the release of bioactive molecules, micro-nano topology can regulate and accelerate the migration and growth of tissues and cells, which greatly improves the biological functionality of nerve grafts, and is more conducive to the long-term treatment and clinical treatment of patients with nerve tissue damage. recovery.

3. The preparation method of the present invention is non-toxic, harmless, environmentally friendly, simple and feasible, easy to operate, the near-infrared illumination time is adjustable, and the activity of cells and tissues and the scientificity of experimental results are not affected during the illumination process. The dosage of photothermal nanoparticles can be flexibly changed according to actual needs to achieve the regulation of different release rates of bioactive molecules. Through electrospinning technology and regulation of biomaterial composition parameters, biomaterial scaffolds with different mechanical properties can be obtained, and biomaterial scaffolds with different mechanical properties can be obtained. Thermally responsive and topologically functionalized personalized tissue-engineered neural grafts to match the clinical needs of different patients, enabling personalized treatments and applications of neural tissue engineering and regenerative medicine.

Description of drawings

Fig. 1 is the modification schematic diagram of the photothermal nanoparticle of the present invention;

2 is a schematic diagram of the topology preparation of the surface of the present invention having anisotropic topology;

3 is a schematic diagram of preparing a biomimetic neural scaffold with biological activity by electrospinning according to the present invention;

FIG. 4 is a schematic diagram of the scanning electron microscope topography of the modified MWCNT of the present invention;

Fig. 5 is the light microscope observation schematic diagram of the surface morphology of the neural scaffold prepared by electrospinning of the present invention;

FIG. 6 is a schematic diagram of fluorescence observation of the growth morphology of Schwann cells of the present invention on the surface of the nerve scaffold.

Detailed ways

In order to make those skilled in the art understand the present invention more comprehensively, the technical solutions of the present invention are further described below with reference to examples. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. In addition, it should be understood that after reading the content taught in the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Specific Example 1

The first step is to prepare a nanoparticle loaded with bioactive molecules with photothermal effect:

Take Tris tris to prepare a Tris solution with pH=8.5, then take Dopa powder and add it to the Tris solution to prepare a polydopamine solution, and finally add 50 mg of nanoparticles to 10 ml of the polydopamine solution. Wrap the container containing the mixed solution with tin foil to protect from light, ventilate, and place it in a mechanical shaker to shake. After shaking, the solution was removed from the shaker container and the supernatant was discarded, and the pellet was dried. The powder obtained by drying was washed again with Milli-Q. After centrifugation again, the supernatant was discarded, and the precipitate was dried to obtain Dopa@MWCNT powder. Add Dopa@MWCNT powder into 20ml DFO/YR mixed solution and shake it in a mechanical shaker. After shaking, remove the solution from the shaker and place it in a centrifuge. Finally, the supernatant was discarded, and the precipitate was dried to obtain YR\DFO-Dopa@MWCNT.

The second step is to prepare a solution of chitosan and Artemisia annua:

Dissolve glacial acetic acid in water to prepare a 3% glacial acetic acid solution. Use 3% glacial acetic acid solution as the solvent of chitosan, add chitosan powder, add 7g chitosan to 100ml 3% glacial acetic acid solution, seal the mouth of the beaker with plastic wrap, and stir on a magnetic stirrer 24h. Then add 2 g Artemisia annua to the chitosan solution, and stir for 24h. The prepared solution of chitosan and Artemisia japonica was stored in a 4°C refrigerator in a sealed manner.

The third step is to prepare an electrospinning receiver plate with anisotropic topology:

A drop of chitosan and Artemisia japonica solution was dropped onto a glass disc with a syringe, and a hydrogel with anisotropic topology and a topological structure width of 30 μm was imprinted on the glass disc with the solution dropped, and allowed to stand at room temperature for 24 hours. After 24 h, the anisotropic topology hydrogel and glass disk were separated. The removed glass disc was placed in a large dish, and alkali-treated with 4% NaOH solution for 15 min. Then carefully wash the glass disc with Milli-Q for more than three times until it is neutral, and then observe it under a light microscope after drying. Cover the cardboard with aluminum foil, which is used to connect the electrodes on the back. A glass disc with anisotropic topology is attached to the front of the aluminum foil board. This receiving board is the receiving end of the electrospinning machine.

The fourth step uses electrospinning technology to prepare a biomimetic tissue engineering scaffold with photothermal responsiveness and controllable drug release:

First, the chitosan artemisia annua electrospinning receiver plate with a topological distance of 10 μm was connected to the electrode, and then the YR\DFO-Dopa@MWCNT-PCL/CS mixed solution (10wt% PCL and 2wt% CS volume ratio was 50:1) was connected to the electrode. ) into the special syringe for electrospinning, electrospinning parameters: voltage 20kV, distance from needle to receiving plate 15cm, pushing speed 0.160ml/h, time 5h. After electrospinning, the YR/DFO-Dopa@MWCNT-PCL/CS scaffold was peeled off from the glass slide in distilled water, and then dried in a drying box to obtain a biomimetic topological tissue engineering with the function of promoting nerve regeneration on the surface. bracket.

Specific embodiment 2

The first step is to prepare a nanoparticle loaded with bioactive molecules with photothermal effect:

Take Tris tris to prepare a Tris solution with pH=8.5, then add Dopa powder to the Tris solution to prepare a polydopamine solution, and finally add 100 mg of nanoparticles to 10 ml of the polydopamine solution. Wrap the container containing the mixed solution with tin foil to protect from light, ventilate, and place it in a mechanical shaker to shake. After shaking, the solution was removed from the shaker container and the supernatant was discarded, and the pellet was dried. The powder obtained by drying was washed again with Milli-Q. After centrifugation again, the supernatant was discarded, and the precipitate was dried to obtain Dopa@MWCNT powder. Add Dopa@MWCNT powder into 20ml DFO/YR mixed solution and shake it in a mechanical shaker. After shaking, remove the solution from the shaker and place it in a centrifuge. Finally, the supernatant was discarded, and the precipitate was dried to obtain YR\DFO-Dopa@MWCNT.

The second step is to prepare a solution of chitosan and Artemisia annua:

Dissolve glacial acetic acid in water to prepare a 3% glacial acetic acid solution. Use 3% glacial acetic acid solution as the solvent of chitosan, add chitosan powder, add 100 ml of 3% glacial acetic acid solution to 3 g of chitosan, seal the mouth of the beaker with plastic wrap, and stir on a magnetic stirrer 24h. Then add 2 g Artemisia annua to the chitosan solution, and stir for 24h. The prepared solution of chitosan and Artemisia japonica was stored in a 4°C refrigerator in a sealed manner.

The third step is to prepare an electrospinning receiver plate with anisotropic topology:

A drop of chitosan and Artemisia japonica solution was dropped on a glass disc with a syringe, and a hydrogel with an anisotropic topology and a topology width of 10 μm was imprinted on the glass disc with the solution dripped, and left at room temperature for 24h. After 24 h, the anisotropic topology hydrogel and glass disk were separated. The removed glass disc was placed in a large dish, and alkali-treated with 4% NaOH solution for 15 min. Then carefully wash the glass disc with Milli-Q for more than three times until it is neutral, and then observe it under a light microscope after drying. Cover the cardboard with aluminum foil, which is used to connect the electrodes on the back. A glass disc with anisotropic topology is attached to the front of the aluminum foil board. This receiving board is the receiving end of the electrospinning machine.

The fourth step uses electrospinning technology to prepare a biomimetic tissue engineering scaffold with photothermal responsiveness and controllable drug release:

First, the chitosan artemisia annua electrospinning receiver plate with a topological distance of 30 μm was connected to the electrode, and then the YR\DFO-Dopa@MWCNT-PCL/CS mixed solution (10wt% PCL and 2wt% CS volume ratio was 25:1) was connected to the electrode. ) into the special syringe for electrospinning, electrospinning parameters: voltage 20kV, distance from needle to receiving plate 15cm, pushing speed 0.160ml/h, time 5h. After electrospinning, the YR/DFO-Dopa@MWCNT-PCL/CS scaffold was peeled off from the glass slide in distilled water, and then dried in a drying box to obtain a biomimetic topological tissue engineering with the function of promoting nerve regeneration on the surface. bracket.

Specific embodiment 3

The first step is to prepare a nanoparticle loaded with bioactive molecules with photothermal effect:

Take Tris tris to prepare a Tris solution with pH=8.5, then add Dopa powder into the Tris solution to prepare a polydopamine solution, and finally add 150 mg of nanoparticles to 10 ml of the polydopamine solution. Wrap the container containing the mixed solution with tin foil to protect from light, ventilate, and place it in a mechanical shaker to shake. After shaking, the solution was removed from the shaker container and the supernatant was discarded, and the pellet was dried. The powder obtained by drying was washed again with Milli-Q. After centrifugation again, the supernatant was discarded, and the precipitate was dried to obtain Dopa@MWCNT powder. Add Dopa@MWCNT powder into 20ml DFO/YR mixed solution and shake it in a mechanical shaker. After shaking, remove the solution from the shaker and place it in a centrifuge. Finally, the supernatant was discarded, and the precipitate was dried to obtain YR\DFO-Dopa@MWCNT.

The second step is to prepare a solution of chitosan and Artemisia annua:

Dissolve glacial acetic acid in water to prepare a 3% glacial acetic acid solution. Use 3% glacial acetic acid solution as the solvent of chitosan, add chitosan powder, add 100 ml of 3% glacial acetic acid solution to 5g chitosan, seal the mouth of the beaker with plastic wrap, and stir on a magnetic stirrer 24h. Then add 2 g Artemisia annua to the chitosan solution, and stir for 24h. The prepared solution of chitosan and Artemisia japonica was stored in a 4°C refrigerator in a sealed manner.

The third step is to prepare an electrospinning receiver plate with anisotropic topology:

A drop of chitosan and Artemisia japonica solution was dropped onto a glass disc with a syringe, and a hydrogel with an anisotropic topology and a topology width of 50 μm was imprinted on the glass disc with the solution dripped, and allowed to stand at room temperature for 24 hours. After 24 h, the anisotropic topology hydrogel and glass disk were separated. The removed glass disc was placed in a large dish, and alkali-treated with 4% NaOH solution for 15 min. Then carefully wash the glass disc with Milli-Q for more than three times until it is neutral, and then observe it under a light microscope after drying. Cover the cardboard with aluminum foil, which is used to connect the electrodes on the back. A glass disc with anisotropic topology is attached to the front of the aluminum foil board. This receiving board is the receiving end of the electrospinning machine.

The fourth step uses electrospinning technology to prepare a biomimetic tissue engineering scaffold with photothermal responsiveness and controllable drug release:

First, the chitosan artemisia annua electrospinning receiver plate with a topological distance of 50 μm was connected to the electrode, and then the YR\DFO-Dopa@MWCNT-PCL/CS mixed solution (10wt% PCL and 2wt% CS volume ratio was 25:1) was connected to the electrode. ) into the special syringe for electrospinning, electrospinning parameters: voltage 20kV, distance from needle to receiving plate 15cm, pushing speed 0.160ml/h, time 5h. After electrospinning, the YR/DFO-Dopa@MWCNT-PCL/CS scaffold was peeled off from the glass slide in distilled water, and then dried in a drying box to obtain a biomimetic topological tissue engineering with the function of promoting nerve regeneration on the surface. bracket.

Specific Example 4

The first step is to prepare a nanoparticle loaded with bioactive molecules with photothermal effect:

(1) First, take Tris to prepare a Tris solution with pH=8.5, then take Dopa powder and add it to the Tris solution to prepare a polydopamine solution, and add nanoparticles to the polydopamine solution

(2) Then wrap the container of the mixed solution with tinfoil to protect from light and ventilate, put it into a mechanical shaker to shake, and after shaking, the solution is discarded from the shaker container and the supernatant is taken out and the precipitate is dried. Then, the powder obtained by drying is washed with Milli-Q. After centrifugation again, the supernatant was discarded, and the precipitate was dried to obtain Dopa@MWCNT powder

(3) Add Dopa@MWCNT powder into the mixed solution of DFO solution and YR solution, put it into a mechanical shaker to shake, and then take out the solution from the shaker and put it into a centrifuge for centrifugation. Finally, discard the supernatant and dry the precipitate to obtain YR\DFO-Dopa@MWCNT

The second step is to prepare a solution of chitosan and Artemisia annua:

(1) The glacial acetic acid was configured into a 3% glacial acetic acid solution.

(2) Take out the chitosan powder, use chitosan as the solute and 3% glacial acetic acid solution as the solvent, put it into a beaker according to the proportion, put it into a rotor, and seal the mouth of the beaker with a plastic wrap to prevent the acetic acid from volatilizing. Put the beaker into a magnetic stirrer and stir for 24h to prepare a 3% chitosan solution.

(3) After 24 hours, take out the stirred solution and take out Artemisia japonica. Add Artemisia seeds to the solution. Continue stirring with a magnetic stirrer for 24 h, and seal the beaker mouth with plastic wrap. Configure into the desired CS+SA solution. Store the prepared solution in a 4°C refrigerator.

The third step is to prepare an electrospinning receiver plate with anisotropic topology:

(1) Inhale the prepared CS+SA solution into a 1ml syringe. Use a syringe to drop a drop of the solution on the glass disc. Then, the anisotropic topology hydrogel was covered on the glass disc to imprint the CS+SA solution, and at the same time, it was ensured that no bubbles were generated in the solution between the anisotropic topology hydrogel and the glass disc. It was then placed in the air and allowed to stand at room temperature for 24h.

(2) After 24 h, the anisotropic topology hydrogel and glass disc were separated. At this point, the CS+SA solution had solidified into an anisotropic topology and was attached to the glass disc.

(3) Put the removed glass disc into a large dish, carefully immerse the glass disc with 4% NaOH solution, soak the glass disc in NaOH solution for half an hour, and then carefully wash the glass disc with triple distilled water for more than three times until The cleaning solution was detected as neutral by pH test paper, and then dried and stored for use to obtain an electrospinning receiving plate with anisotropic topology.

The fourth step uses electrospinning technology to prepare a biomimetic tissue engineering scaffold with photothermal responsiveness and controllable drug release:

(1) Prepare aluminum foil and cardboard, cover the aluminum foil and wrap the cardboard, the aluminum foil plate is the receiving plate, take out the preserved glass wafer with anisotropic topology, and place the side with anisotropic topology outward. , the back of the glass slide is adhered to the surface of the receiving plate, that is, the electrospinning receiving plate is completed and can be directly used as the receiving end of the electrospinning machine.

(2) 10wt% PCL was prepared, the mass volume ratio of PCL and hexafluoroisopropanol was 1:10, and the mixture was shaken and stirred for 1 d. Then configure 2wt% CS, the mass volume ratio of CS and hexafluoroisopropanol is 1:50, and take out after shaking and stirring for 1 d.

(3) Mix 2wt% CS and 10wt% PCL at room temperature, the ratio is 5ml 10wt% PCL and 200μL 2%wtCS, put it on a shaker for shaking, and take it out after shaking and stirring for 1 d.

(4) Mix and stir the prepared PCL/CS solution with YR\DFO-Dopa@MWCNT powder to ensure that it is fully stirred

(5) In the ultra-clean bench, the receiving plate with electrospinning with anisotropic topology is placed on the receiving end of the electrospinning machine, and the receiving plate is connected to the electrode; at the same time, the above mixed solvent is injected into the electrospinning machine In the special syringe, it is the transmitting end of the electrospinning machine. At this time, the transmitting end and the receiving end of the electrospinning machine have been assembled. After adjusting the advancing speed of the transmitting end and confirming that the entire machine is assembled correctly, pull down the protective cover of the clean bench. , turn on the power to start the electrospinning process.

(6) Turn off the instrument after electrospinning for 5 hours, remove the spun receiving plate with the bionic topological tissue engineering scaffold, and peel off the scaffold and receiving plate in a glass dish with distilled water. During the peeling process, the topological structures already present on the scaffolds are destroyed or deformed. The peeled scaffold was placed on a transparent round glass slide placed in a glass dish in advance. The scaffold is taken out of the water, dried and stored at room temperature to obtain a tissue engineering scaffold with bioactive molecules that promote nerve regeneration in the scaffold and a biomimetic topology structure on the scaffold surface.

Claims (9)

1. a preparation method of a biomimetic tissue engineering scaffold with photothermal responsive controllable drug release, the scaffold body material is a macromolecular biomaterial with excellent biocompatibility; it is characterized in that: the scaffold comprises Controllable release bioactive molecules with the function of promoting nerve regeneration, the bioactive molecules are loaded by nanoparticles with photothermal effect; the surface of the scaffold has an anisotropic topology that can regulate cell-oriented growth and migration; The scaffold can better bionic nerve regeneration microenvironment; The preparation method of the biomimetic tissue engineering scaffold with photothermal responsiveness and controllable drug release comprises the following steps: (1) Preparation of bioactive nanoparticles loaded with bioactive molecules that promote nerve regeneration; (2) Preparation of polymer biomaterial solution; (3) Micro-molding is used to prepare an electrospinning receiving substrate with an anisotropic topology on the surface; (4) The biomimetic tissue engineering scaffold with photothermal responsive controllable drug release is prepared by electrospinning (1) and (2) onto the receiving substrate of (3) after blending. 2. The method for preparing a biomimetic tissue engineering scaffold with photothermal responsiveness and controllable drug release as claimed in claim 1, wherein the polymer biomaterial with excellent biocompatibility is chitin, Chitosan, alginate, collagen, polycaprolactone (PCL) or polylactide (PLA). 3. The method for preparing a biomimetic tissue engineering scaffold with photothermal responsiveness and controllable drug release as claimed in claim 1, wherein the controllable release bioactive molecule for promoting nerve regeneration is polysaccharide, Nucleic acids, proteins, lipids, polypeptides or growth factors. 4. The preparation method of a biomimetic tissue engineering scaffold with photothermal response controllable drug release as claimed in claim 1, wherein the nanoparticle with photothermal effect is carbon nanotube, ferric tetroxide Or gold nanoparticles. 5. The method for preparing a biomimetic tissue engineering scaffold with photothermal responsiveness and controllable drug release as claimed in claim 1, wherein the anisotropic topological structure capable of regulating cell-oriented growth and migration comprises: Oriented nanotopology, regularly arranged microgrooves, or a combination of the two exist. 6. The preparation method of a biomimetic tissue engineering scaffold with photothermal responsiveness and controllable drug release as claimed in claim 1, wherein the scaffold can better be a microenvironment for biomimetic nerve regeneration, including physical microenvironments. Environment, glial cell microenvironment, nerve growth factor microenvironment, extracellular matrix microenvironment. 7. The preparation method of a biomimetic tissue engineering scaffold with photothermal responsive controllable drug release as claimed in claim 1, characterized in that: the method for modifying photothermal nanoparticles in the step (1): can Use dopamine hydrochloride (DOPA), genipin, or aminopropyl ethoxysilane coupling agent (APTE) to coat and modify the photothermal nanoparticles; in the step (2), the polymer solution can be a good biological phase Compatible natural or synthetic polymer solutions. 8. The preparation method of the biomimetic tissue engineering scaffold with photothermal responsiveness controllable drug release as claimed in claim 1, characterized in that: in the step (3), the mold used in the micro-molding method comprises a PDMS mold or a PMMA mold Mold; the electrospinning receiving substrate can be a natural biomaterial or a synthetic biomaterial substrate, and the substrate is formed by imprinting on a cover glass by a micro-molding method. 9 . The method for preparing a biomimetic tissue engineering scaffold with photothermal responsiveness and controllable drug release according to claim 1 , wherein in the step (4), the biologically active nanoparticles are co-coated with the polymer biomaterial solution. 10 . The ratio of mixing mass to volume ratio is 1:100 to 1:10000.

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